Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex-sorted Frozen-thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

甘蓝黑腐病菌rPf基因簇对胞外蛋白酶Ⅰ基因表达的调控

甘蓝黑腐病黄单胞菌（ＸａｎｔｈｏｍｏｎａｓｃａｍｐｅｓｔｒｉｓＰｖ．ｃａｍｐｅｓｔｒｉｓ）产生的胞外蛋白酶Ⅰ在致病的早期阶段起重要作用，该酶以及其它胞外酶和胞外多糖的合成受一致病因子调控基因簇（ｒＰｆ基因簇）的正向调控。本研究利用带有β－半乳糖苷酶报道基因（ｌａｃＺ）的转座子Ｔｎ５－Ｂ２０诱变蛋白酶Ⅰ基因克隆，获得了ｌａｃＺ在蛋白酶Ⅰ基因启动子控制下表达的Ｔｎ５－Ｂ２０插入突变质粒。通过将这种突变质粒导入野生型和各ｒｐｆ基因突变体菌株后，测定ｌａｃＺ基因在细胞生长周期中的表达水平，不仅进一步证实了这些ｒｐｆ基因对蛋白酶Ⅰ基因的正向调控作用，而且明确了它们的调控水平．发现ｒｐｆＡ、ｒｐｆＣ、ｒｐｆＥ、ｒｐｆＧ或ｒｐｆＨ突变后，蛋白酶Ⅰ基因的转录会降低９０％左右，而ｒｐｆＢ突变后，蛋白酶Ⅰ基因的转录只降低４８％。     

High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

Effects of Water Hardness and Calcium: Magnesium Ratios on Reproductive Performance and Offspring Quality of Macrobrachium rosenbergii

This study evaluated the effect of specific calcium and magnesium ratios at two hardness values on reproductive performance and offspring quality of Macrobrachium rosenbergii brood‐females. Hatchery water containing 110.5 mg/L CaCO3 hardness was considered baseline as it has been used successfully to hatch and rear larvae and was used as the control treatment. At each hardness value of 150 and 190 mg/L CaCO₃, four experimental water treatments were made in triplicate. Each treatment was adjusted using soluble salts (CaCl₂·H₂O, MgSO₄·7H₂O, and MgCl₂·6H₂O) to provide the hardness and calcium to magnesium ratios of 0:20, 20:80, 50:50, and 80:20 needed. Each of the experimental tanks (140 L) were stocked with six females and one male. The results indicated that both hardness and Ca:Mg ratios affect brood‐female reproductive qualitative parameters such as intermolt period, egg hatchability, egg dry weight, and egg‐clutch somatic index (ESI) parameters (P < 0.05) but not fecundity and eggs per spawn. The results revealed that brood‐females at 150 mg/L hardness showed greater reproductive performance than at 190 and 110.5 mg/L hardness. The findings also demonstrated that the treatment 50Ca50Mg at 150 mg/L hardness with 38.8 mg/L calcium and 12.9 mg/L magnesium had optimum reproductive performance and offspring quality for M. rosenbergii brood‐females.     

Fluoroquinolone levels in healthy dog urine following a 20‐mg/kg oral dose of enrofloxacin exceed mutant prevention concentration targets against Escherichia coli isolated from canine urinary tract infections

A 3‐day course of oral enrofloxacin is effective for treating uncomplicated urinary tract infection (UTI) in dogs when administered 20 mg/kg Q24H. However, emergence of fluoroquinolone‐resistant mutants of uropathogens is a concern. Urine concentrations of enrofloxacin and ciprofloxacin were measured in six healthy dogs following dose of enrofloxacin 20 mg/kg. Mutant prevention concentrations of Escherichia coli isolated from canine UTI were also determined against ciprofloxacin. Urine AUC(24)/MPC ratios considering ciprofloxacin concentrations ranged 3819–7767, indicating that selection of resistant E. coli mutants in dogs with uncomplicated UTIs is unlikely in the bladder given that an AUC(24)/MPC = 39 is considered to be protective against mutant selection for ciprofloxacin. However, additional studies are required to evaluate the effects of this enrofloxacin treatment protocol on bacteria that colonize anatomic sites where fluoroquinolones achieve lower concentrations compared to the urinary bladder.     

Compensatory Growth of Pond-reared Hybrid Striped Bass, Morone chrysops × Morone saxatilis, Fingerlings

Compensatory growth (CG) or “catch‐up growth” is a period of super‐accelerated growth following a period of suboptimal conditions (i.e., lack of prey availability or overwintering). Little is known about the CG response in pond‐raised fish and whether hybrid striped bass (HSB), Morone chrysops × Morone saxatilis, might exhibit the rapid growth states or improvements in other production characteristics that may accompany the response. To evaluate the potential for CG in HSB culture, a 16‐wk growth trial in twelve 0.1‐ha earthen ponds was conducted. Approximately 2850 fish (mean weight ± SD = 3.2 g ± 1.1) were stocked into ponds and subjected to one of four cyclic feeding regimens. Treatment regimens included a control (0 wk, fed twice daily to apparent satiation) and cycles of 1, 2, or 4 wk of feed deprivation, followed by 1, 2, or 4 wk of feeding to apparent satiation. Fish in the 4‐wk feeding regimen were offered feed twice during the feed‐deprivation period (once every other week). Growth, specific growth rate (SGR), hepatosomatic index (HSI), intraperitoneal fat ratio, and condition factor (CF) were measured every other week, while overall growth, feed efficiency (FE) (FE = [weight gain/feed fed] × 100), and survival were calculated at the trial termination. The effect of these feeding regimens on water quality was examined by monitoring pH, turbidity, total ammonia nitrogen, nitrite–nitrogen, nitrate–nitrogen, soluble reactive phosphorus, and chlorophyll a weekly; total nitrogen and phosphorous biweekly; and dissolved oxygen and temperature twice daily. Cyclic feeding elicited CG; fish subjected to the 2‐wk regimen had a significantly higher SGR than 0‐wk controls during all but the final refeeding period. FE was higher for all fish in the cyclic regimens, although only FE for fish in the 2‐wk regimen was statistically greater (40%) than the controls. HSI was the most responsive measure and significantly decreased in the 2‐ and 4‐wk treatments during feed‐deprivation period and overcompensated during the refeeding period. CF also varied with feeding cycle and proved to be an effective nonlethal measure of predicting a CG response. No statistical differences in water quality parameters were observed. These data suggest that CG can be effectively induced in pond‐raised HSB and that the increase in FE warrants further research for practical application. Future pond studies with fingerling HSB fish should be conducted with emphasis on feed‐deprivation periods of 2 wk and refeeding periods of at least twice that of the feed‐deprivation period.     

Environmental Manipulation of Growth and Sexual Maturation in Yellow Perch, Perca flavescens

Yellow perch, Perca flavescens, exhibit sexually dimorphic growth detected as the fish enter puberty. The purpose of this study was to evaluate the differential influences of photoperiod and temperature on the sexual maturation and dimorphic growth pattern of yellow perch. Four combinations of constant (CP) or natural (NP) photoperiod with constant (CT) or natural (NT) temperature were evaluated (CPCT, CPNT, NPCT, NPNT). Following 6 months of growth, results indicate that (1) CT is the strongest promoter of overall growth regardless of photoperiod. Perch reared under CT were 26.4 ± 8.1 g heavier than perch reared under NT (P < 0.0001). (2) CP also promotes overall growth regardless of temperature. CP perch outgrew NP perch by 7.0 ± 8.1 g (P < 0.0872). (3) NP (decreasing) initiates maturation in yellow perch regardless of temperature. (4) NT is required for late maturational processes. While both NP groups initiated maturation, only NPNT females were able to recruit oocytes to a vitellogenic stage. Only NPNT males were able to sustain spermiation to the end of the study. (5) CPCT confer the best overall growth performance, fully inhibit maturation in both genders, and suppress a sexually dimorphic growth pattern up to 53 g average weight. These results indicate that using a CPCT regime during intensive fingerling production will produce a larger, more uniform fish population that could alleviate losses as a result of cannibalism. Perch that remained immature did not exhibit a dimorphic growth pattern.